The overall goal of our research is to understand the pathogenicity of Streptococcus pneumoniae, the most common cause of bacteremia, bacterial meningitis, otitis media and community-acquired pneumonia in the U.S.A. Current capsule-based vaccines, which only contain a subset of the capsular types in circulation, do not provide adequate protection from pneumonia and otitis media, which account for the majority of S. pneumoniae morbidity. Despite more than a century of research, understanding of S. pneumoniae virulence factors is limited. Furthermore, almost nothing is known concerning the regulation of S. pneumoniae virulence factors during infection. These limitations, plus an increasing incidence of antibiotic-resistance mandate increased study of the pathogenicity of this organism. We have completed a large-scale screen that resulted in the identification of 233 genes that are essential in a murine model of pneumonia. Additionally, we tested the importance of each of these genes in murine models of bacteremia and nasopharygeal carriage. Included among these novel virulence factors are 21 surface proteins, and 20 putative regulators that we hypothesize coordinate tissue-specific virulence gene expression. The first goal of the proposed work is to gain an understanding of both the regulation and mechanisms of action of two factors hypothesized to localize to the bacterial surface and interact with host components. Mutational analyses and virulence assays will be combined to define their interacting domains. The second goal is to identify major virulence gene regulons and their modes of coordination during infection. Five putative transcription factors identified in our screen will be placed under inducible expression, and the subset of genes regulated by each will be determined by transcriptional profiling on microarrays. For selected genes, the level of expression during nasopharyngeal carriage in mice and humans will be determined using quantitative RT-PCR. The requirement for each cognate regulator for expression of these virulence genes during infection of mice will be confirmed. These studies will enhance our knowledge and understanding of S. pneumoniae-host interactions and virulence mechanisms, and will constitute the first broad study of S. pneumoniae virulence gene regulation. These studies will aid in the development of novel vaccines, and will suggest new targets for antimicrobial drug development.